Synthesis, Characterization, and Bioconjugation of Fluorescent Gold

Cheng-An J. Lin,†,¶ Ting-Ya Yang,†,¶ Chih-Hsien Lee,†,¶ Sherry H. Huang,†,¶ Ralph A. Sperling,‡ Marco Zanella,‡ Jimmy K. Li,†,¶,# Ji-Lin Shen,§ Hsueh-Hsiao Wang,⬜ Hung-I Yeh,⬜ Wolfgang J. Parak,‡,* and Walter H. Chang†,¶,* †

Department of Biomedical Engineering, Chung Yuan Christian University, Chung-Li 32023, Taiwan, ROC, ‡Fachbereich Physik, Philipps Universita¨t Marburg, Renthof 7, 35037 Marburg, Germany, §Department of Physics, Chung Yuan Christian University, Chung-Li 32023, Taiwan, ROC, ⬜Cardiac Medicine, Mackay Memorial Hospital, Taipei, Taiwan, ROC, and ¶R&D Center for Membrane Technology, Center for Nano Bioengineering, Chung Yuan Christian University, Chung-Li 32023, Taiwan, ROC. # Present address: Harvard⫺MIT Health Sciences and Technology, Brigham and Women‘s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139.

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oble metal nanoclusters (NCs) such as Au and Ag with sizes below 2 nm have gained great attention in the past decade. Focus of interest ranges from fundamental properties such as photoluminescence,1⫺8 optical chirality,9⫺12 ferromagnetism,13 and redoxlike and quantized double layer charging behavior14⫺17 to potential applications such as single molecule optoelectronics,18,19 sensing,20 and bioassays.21,22 This is based on the particular size-dependent properties of the clusters. They can be considered as intermediates between atomic and nanoparticle (⬎2 nm) behavior.23 Gold nanoparticles (NPs) show a size-dependent plasmon absorption band when their conduction electrons are confined to dimensions smaller than the electron mean free pathlength (ca. 20 nm).24 However, Au nanoparticles smaller than 2 nm (which we term Au nanoclusters) no longer possess plasmon resonance and Mie’s theory no longer can be applied.25⫺27 In fact small metal NCs have sizes comparable to the Fermi wavelength of electrons (ca. 0.7 nm), which results in molecule-like properties including sizedependent fluorescence20,23,28 and discrete size-dependent electronic states.14,29⫺31 So far there are only few studies about the fluorescence of metal NCs,32⫺34 though photoluminescence (PL) of noble metals is a known phenomenon. First reports are based on bulk metals,35 but soon quasi-size dependence was discovered by the fact that the PL is enhanced on roughened surwww.acsnano.org

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Synthesis, Characterization, and Bioconjugation of Fluorescent Gold Nanoclusters toward Biological Labeling Applications

ABSTRACT Synthesis of ultrasmall water-soluble fluorescent gold nanoclusters is reported. The clusters have

a decent quantum yield, high colloidal stability, and can be readily conjugated with biological molecules. Specific staining of cells and nonspecific uptake by living cells is demonstrated. KEYWORDS: colloidal gold · fluorescent nanoparticles · nanoclusters

faces36 (i.e., at conditions with reduced radius of curvature). The fluorescent properties of Au NCs make them potential labels for biologically motivated experiments.37 Compared to commonly used CdSe/ZnS semiconductor NPs (so-called quantum dots), which possess size-dependent fluorescence in cases where the particle size is smaller than the exciton Bohr radius (about 4⫺5 nm for CdSe),38 Au NCs do not contain toxic heavy metals.39 Though depending on their size also, Au NCs can have cytotoxic effects, for example by binding to DNA40 they are a potential alternative to quantum dots as fluorescence labels. In this work we describe an easy synthesis for water-soluble fluorescent Au NCs and compare their performance in terms of colloidal and photophysical properties to the one of colloidal CdSe/ ZnS NPs. The focus hereby is set on a detailed characterization of practically important parameters rather than on understanding the underlying mechanisms. RESULTS AND DISCUSSION Photophysical Characterization. Synthesis of Au NCs was based on precursor-induced Au NP etching in organic phase and ligand

*Address correspondence to [email protected], [email protected]. Received for review September 29, 2008 and accepted December 31, 2008. Published online January 27, 2009. 10.1021/nn800632j CCC: $40.75 © 2009 American Chemical Society

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most organic fluorophores and quantum dots, it is already in the range needed for applications in optical devices and biosensors. Impurities are always problematic for working with colloidal particles, as they can coexist in the solution so that the optical properties detected from this solution comprise both, the particles and the impurities. As will be explained in the following sections we were able to separate particles from potential impurities such as excess ligands or precursors by size exclusion chromatography and gel electrophoresis, and the presence of the NCs in the purified solutions was verified with TEM. In this way it was assured that the photoluminescence originates from the NCs. Photoluminescence of Au NCs has been reported before and several models have been proposed to explain this photoluminescence mechanism. Though this study does not contribute in understanding the Figure 1. (a) General strategy to fabricate water-soluble fluorescent Au nanoclusters. origin and mechanism of photoluminesDDAB-stabilized gold nanoparticles (AuNP@DDAB) are etched by the addition of Au precursors (HAuCl4 or AuCl3) to smaller nanoclusters (AuNC@DDAB). By the addition of reduc- cence of Au NCs there is work by other ing agent (TBAB) the Au NCs grow again reversibly into bigger Au NPs. The hydrophobic groups in this direction. A theoretical study AuNC@DDAB become water soluble upon ligand exchange with dihydrolipoic acid (AuNC@DHLA); (b) TEM images of AuNP@DDAB, AuNC@DDAB, and AuNC@DHLA; 100 par- explains photoluminescence of nanosized noble metals by electronic transitions beticles were randomly selected for measuring the size distribution, resulting in 5.55 ⴞ 0.68 nm, 3.17 ⴞ 0.35 nm, and 1.56 ⴞ 0.3 nm in diameter, respectively. (c) Pictures of particle so- tween occupied d bands and states above lutions under daylight. Contrary to AuNP@DDAB solution which features the red color of 29 surface plasmon absorption, AuNC@DDAB and AuNC@DHLA display a colorless and brown the Fermi level (usually the sp bands). Whetten et al. attribute photoluminestranslucent solution without plasmon absorption, respectively. (d) Pictures of the same particle solutions under UV excitation. The AuNC@DHLA solution shows red photolumines- cence in the red/near-infrared (NIR) of Au cence. NCs comprising 28 Au atoms capped with glutathione to the recombination between exchange with reduced lipoic acid (DHLA, dihydrolithe ground-state and two distinctively different expoic acid) to transfer the particles into aqueous solucited states, that is, an intraband and interband transition. Only after phase transfer to aqueous solution do the NCs exhibit a pronounced red photoluminescence. tion.5 Murray et al. associated the photoluminescence to interband transitions between the filled 5d10 band and Exposure of the AuNC@DHLA particle solution to UV light (365 nm) led to an increase in photoluminescence the 6(sp)1 conduction band2 or to participation of localized core surface states.43 Other possible mechanisms (photobrightening). Photoluminescence (PL) and phohave been suggested such as ligand-to-metal charge toluminescence excitation (PLE) spectra of the cluster solution are shown in Figure 2. The nonshifted emission transfer (LMCT) which is mainly affected by the types of ligands,20 and ligand-to-metal⫺metal charge transpeak via changing the excitation wavelength gives strong evidence that the observed emission is a real lu- fer (LMMCT) which is commonly referred to as aurominescence from the relaxed states rather than scatter- philic interaction.44 Also fluorescence emission of larger ing effects. Using Rhodamine 6G (Q ⫽ 0.95 in ethanol) gold nanoparticles has be reported recently.45 as the reference, the quantum yield (Q) of AuNC@DHLA In an additional experiment photostability of differwas determined to be 3.45 ⫾ 0.41% in methanol and ent water-soluble fluorophores was compared (for data 1.83 ⫾ 0.32% in water (pH 9). This is 8 orders of magni- see the Supporting Information). The fluorescent gold tude higher with respect to the quantum yield renanoclusters (AuNC@DHLA) as synthesized here exhibported for bulk gold (Q ⫽ 10⫺10),35 approximately 1⬃2 ited less photobleaching than organic fluorophores orders of magnitude higher than the quantum yield of (fluorescein, rhodamine 6G). However, AuNC@DHLA Au NCs derived with other synthesis routes (Q ⫽ 10⫺3 was more prone to photobleaching than semiconduc⫺10⫺6),1,2,5,30,41,42 and comparable to the quantum yield tor quantum dots (CdSe/ZnS), which show an excellent recently reported by Huang et al. for similar Au NCs.20 photostability. Though the quantum yield of the AuNC@DHLA nanoColloidal Characterization. To purify the NCs from evenclusters is still 1 order of magnitude lower than that of tual residual impurities, size exclusion chromatography VOL. 3 ▪ NO. 2 ▪ LIN ET AL.

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and gel electrophoresis was applied. These methods fractionate the initial particle solution and only the desired fraction containing the particles is extracted from the elution profile. As both methods are sensitive to size (gel electrophoresis is in addition sensitive to charge) also estimates of size of the NCs can be extracted from the time-resolved elution profiles.46 The hydrodynamic diameter deff of AuNC@DHLA nanoclusters was determined with size exclusion chromatography to be 1.3 ⬍ deff ⬍ 3.4 nm using 1⫺3 kDa polyethylene glycol (PEG) molecules as size standard46 (Figure 3a). Similarly the hydrodynamic diameter of AuNC@DHLA was determined with gel electrophoresis to be smaller than 5 nm (Figure 3b), using bis(p-sulfonatophenyl)phenylphosphine capped Au NPs of different size as standard. Hereby similar negative charge on the reference Au NPs and the Au NCs reported in this study was assumed.46 These data suggest very small effective particle diameters in colloidal solution well below 5 nm. The size increment compared to the TEM results which refer to the diameter of only the inorganic Au core is at- Figure 2. (a) UVⴚvis absorption spectra of the as-prepared gold nanoparticles (AuNP@DDAB), after etching (AuNC@DDAB), and after ligand tributed to the thickness of the organic capping of the exchange (AuNC@DHLA). The AuNP@DHLA sample was obtained by particle surface with DHLA and adsorbed counterions. ligand exchange from the original AuNP@DDAB sample without etchDue to their small size the AuNC@DHLA particles also ing step. Au samples with DDAB capping were dissolved in toluene, sample with DHLA capping in aqueous solution. (b) Photoluminescence showed excellent colloidal stability in buffer solution (PL, dashed) and photoluminescence excitation spectra (PLE, solid) of such as with 150 mM NaCl. AuNC@DHLA particles. For the PL spectrum, excitation at 490 nm was (Bio-)conjugation. Polyethylene glycol (PEG, 5 kDa) used. For the PLE spectrum, a fixed emission at 650 nm was used. was chosen as the first example of a molecule to conjuattachment of more PEG leads to complete retardation gate to the surface of AuNC@DHLA. PEG-coated particles are known for excellent biocompatibility, reduced on the gel and conjugates with higher stoichiometry could not be resolved. PEGylation extraordinarily imnonspecific interaction with biological molecules and 47,48 proved the stability of AuNC@DHLA in salt-containing cells, and increased coloidal stability in a salt49⫺52 buffer (1 M NaCl) as well as in acidic buffer (pH ⬍ 7). containing solution because of steric repulsion. In 53,54 Similarly also biotin-PEG-amine could be attached to previous studies we have described that the more PEG molecules are attached to nanoparticles, the slower AuNC@DHLA following the same conjugation chemisthe PEGylated nanoparticles run with gel electrophore- try. In this case the unbound end of the PEG containing sis because of increased size of the conjugates. Attach- the biotin moiety is available as an anchor point for further bioconjugation, and the first discrete band can be ment of single amino-terminated PEG molecules ascribed to Au NCs with exactly one biotin functionality (methoxy-PEG-NH2, Mw ⱖ 5000 g/mol) to nanopartiper NC. In a control experiment biotinylation of cles using EDC chemistry can yield to distinctly shifted bands upon gel electrophoresis.53 Here we demonstrate that PEG-NH2 can also be covalently attach to fluorescent AuNC@DHLA particles via EDC, which forms an amine-reactive O-acylisourea intermediate with carboxyl groups and thus mediates amide formation with primary amines. The results shown in Figure 4 indicate that EDC-mediated attachFigure 3. (a) Elution profile of size exclusion chromatography (Sephacryl S-200 gel) ment of PEG-NH2 retards the band of the conjugates on the gel. At least AuNC@DHLA particles versus PEG standards (1000, 3000, 10000 g/mol) as recorded via absorption or refractive index measurements. The PEG standards correspond to theoretically estione discrete retarded band exists mated diameters of 6.6, 3.4, and 1.8 nm. (b) Electrophoretic mobility of Au particles in 2% agawhich we ascribe to Au NCs with ex- rose gels (7.5 V/cm electric field, 20 min running time). Phosphine stabilized Au NPs of 10 and 5 nm core diameter were run in lanes a and b as control. In lanes c and d AuNP@DHLA and actly one PEG attached per parAuNC@DHLA solutions are run. The AuNP@DHLA corresponds to AuNP@DDAB after ligand ex54 ticle. As the Au NCs are very small, change. Images were recorded at daylight (left) and under UV excitation (right).

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AuNC@DHLA was proven by cutting out the biotinylated Au NCs from gel, incubating them with streptavidin, and observation of the resulting increase in diameter due to biotin⫺streptavidin bond formation with gel electrophoresis (for date see the Supporting Information). PEGylation of AnNC@DHLA did not lead to reduction in photoluminescence intensity. Avidin, a glycoprotein was chosen as second example for direct conjugation to AuNC@DHLA, as avidin⫺ biotin technology has been widely used in biomedical research. Avidin molecules contain NH2 groups and therefore can be conjugated to fluorescent AuNC@DHLA by EDC chemistry. Again attachment of avidin led to a reduction in mobility of the conjugates due to its high molecular weight of approximately 66 kDa (see Supporting Information for data). Similar results were obtained for streptavidin. In both cases Figure 4. PEGylation and biotinylation of fluorescent Au nanoclusters (AuNC@DHLA) as probed with conjugation of AuNC@DHLA gel electrophoresis. The COOH groups present on the surface of AuNC@DHLA due to the DHLA capdid not lead to a reduction in ping allow for covalent linkage of NH2-modified PEG molecules via EDC activation: (a) addition of EDC only to AuNC@DHLA serves as negative control without major effect in mobility; (b) addition of EDC un- particle photoluminescence. der the presence of PEGⴚNH2 retards the particle bands; (c) addition of EDC under the presence of Specific Labeling of Fixed Cells. biotinⴚPEGⴚNH2 retards the particle bands. The terminal biotin groups are anchor points for further Biotin, a vitamin essential for attachment of biological molecules via streptavidin. In both cases retardation is caused by linkage of metabolism, is widely distribPEG to the Au particles and the corresponding increment in size. The more the PEG is attached to AuNC@DHLA the larger the conjugates and thus the retardation become. We ascribe the first discrete uted in the body. Significant band to Au particles with exactly one PEG linked per particle. The negative control (lane “N”) represent amounts of endogenous biotin AuNC@DHLA with PEG in the absence of EDC, and lane “C” is a control with AuNC@DHLA only. have been detected in rat kidney, liver, and brain.55 Probing the intracellular distribution of endogenous biotin within liver cells with streptavidinconjugated AuNC@DHLA was therefore used as an example for confirming the functionality of streptavidinconjugation. Human hepatoma cells (HepG2) were fixed with 2% paraformaldehyde and stained with streptavidinconjugated AuNC@DHLA. Unconjugated, PEG-conjugated, and BSAconjugated AuNC@DHLA were used as Figure 5. Labeling of endogenous biotin within human hepatoma cells (HepG2) with fluonegative control, and FITC-avidin was rescent Au NCs conjugated to streptavidin (streptavidin-conjugated AuNC@DHLA). Three negative controls were used: unconjugated AuNC@DHLA, PEG-conjugated AuNC@DHLA, and used as positive control (Figure 5). BoBSA-conjugated AuNC@DHLA. Streptavidin-conjugated FITC served as positive control. All vine serum albumine (BSA) served images were recorded with a 20ⴛ objective (PLAN NEOFLUAR) with a Zeiss confocal microhereby as control for a protein which scope. The fluorescence images were recorded with the following filter sets: AuNC@DHLA:LP does not specifically bind to biotin. How585 em/488 ex, FITC:LP 530/488 ex. The scale bars indicate 50 ␮m. VOL. 3 ▪ NO. 2 ▪ LIN ET AL.

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Figure 6. Nonspecific uptake of unconjugated fluorescent Au nanoclusters (AuNC@DHLA) by human aortic endothelial cells. Cell nuclei were stained with (Hochest 33258) to yield blue fluorescence. The red fluorescence corresponds to the Au NCs: (a) control without Au NCs; (b) serum-supplemented cell medium with 50 nM AuNC@DHLA added; (c) serum-free cell medium with 50 nM AuNC@DHLA added for 5 h.

ever, some red fluorescence can be observed in the labeling of the cells incubated with unconjugated and with BSA-conjugated Au NCs. In both cases this is referred to as nonspecific background. Conjugation of the Au NCs with PEG-reduced nonspecific binding was as expected, which can be seen in the very low red fluorescence intensity. Streptavidin-conjugated Au NCs on the other hand stained the biotin containing cells with high intensity. These results are in agreement with the positive control in which streptavidin-coated FITC was used, which is a common way to label the endogenous biotin.55 The conclusion from in situ observation within liver cells was that the streptavidin-conjugated AuNC@DHLA can specifically label endogeneous biotin. Nonspecific Uptake by Living Cells. In addition preliminary data indicate that fluorescent AuNC@DHLA without any modification did not cause any acute toxicity. Au NCs were nonspecifically incorporated by cells, as it is known also for colloidal quantum dots.56 Uptake of un-

SYNTHESIS Synthesis of Au NCs was based on precursor-induced Au NP etching. First 6-nm diameter gold NPs stabilized with didodecyldimethylammonium bromide (AuNP@DDAB) were synthesized in toluene via an established single-phase reaction58 (experimental details can be found in the Supporting Information). The Au NPs isolated by methanol-induced agglomeration were then re-dissolved in toluene. The resulting red translucent NP solution showed the typical plasmon absorption peak at 520 nm, and transmission electron microscopy (TEM) analysis revealed a mean particle diameter of the inorganic Au core of 5.55 ⫾ 0.68 nm. Subsequent further dropwise addition of gold precursor solution (AuCl3 or HAuCl4 in DDAB-toluene solution) caused a gradual loss of plasmon absorption until the solution turned colorless transparent (see Figure 1). TEM analysis of the transparent solution discovered the presence of small NCs with homogeneous size distribution (mean diameter 3.17 ⫾ 0.35 nm as determined with TEM). This suggests that continuous addition of precursor started to etch the original nanoparticles of around 6 nm diameter (AuNP@DDAB) into smaller nanoclusters of around 3 nm diameter (AuNC@DDAB)20,59 (for a set of timedependent absorption spectra see the Supporting Information). As no change in solvent was involved in the etching procedure the Au NCs at this step still were dispersed in toluene. However, because for intended biological applications water-soluble Au NCs are required, a phase transfer to aqueous solution was carried out. For this purpose ligand exchange with reduced lipoic acid (DHLA, dihydrolipoic acid) was performed, a capping ligand which has already been successfully used for phase transfer.60 Li-

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CONCLUSIONS In this report we describe the synthesis of water-soluble fluorescent gold nanoclusters capped with dihydrolipoic acid (DHLA). The resulting AuNC@DHLA particles have a quantum yield of around 1⫺3%, reduced photobleaching compared to organic fluorophores, and very good colloidal stability. AuNC@DHLA can be conjugated with EDC chemistry to biologically relevant molecules such as PEG, BSA, avidin, and streptavidin. Uptake of AuNC@DHLA by cells did not cause acute toxicity. By their small hydrodynamic diameter (⬍5 nm) and inert nature fluorescent Au NCs might become an interesting alternative to colloidal quantum dots, in particular for applications in which the size and biocompatibility of the label is critical. The weakest point so far remains the relatively low quantum yield. On the basis of the current report as well as on previous breakthrough findings5,8,14,23,34,57 we believe that fluorescent gold nanoclusters have a great potential for applications to biomedical research to offer.

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conjugated AuNC@DHLA was observed in living endothelial cells after around 5 h incubation (Figure 6). Here the uptake efficiency depended significantly on the culture medium, as incorporation was stronger in serum-free than in serumsupplemented medium.

poic acid was freshly reduced by tetrabutylammonium borohydride (TBAB) with a molar ratio of lipoic acid to of TBAB ⫽ 4:1 and added to the AuNC@DDAB particles dispersed in toluene. This led to dark-brown particle agglomerates in the resulting mixture. Particles were precipitated by centrifugation, the toluene-based supernatant was discarded and the remaining particle precipitate could be dispersed in alkaline aqueous solution (pH 9) or methanol (basified by a small amount of alkaline buffer). Further purification was achieved by two runs of ultracentrifugation (110000 rpm) for removing excess DHLA, and the particle solution was concentrated with a centrifuge filter of 30 kDa MWCO (molecular weight cutoff), leading to a colloidally stable transparent solution of NCs without plasmon peak. Successful transfer from toluene to alkaline buffer solution suggests that the added DHLA had replaced the DDAB surfactants driven by the formation of gold-thiol bonds. TEM images showed that the resulting AuNC@DHLA particles were well-dispersed and had an average core diameter of 1.56 ⫾ 0.3 nm. This demonstrates that the diameter of the Au NCs was further decreased during the ligand exchange procedure.

Acknowledgment. This project was supported by the National Science Council (NSC 96-2320-B-033; NSC 97-2627-B-033), the Ministry of Education from Taiwan, and in part by the Deutsche Forschungsgesellschaft (DFG, grant PA794/4-1) and the Bundesministerium fu¨r Bildung and Forschung (BMBF, grant 13N9192).

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Supporting Information Available: Synthesis, size characterization, optical properties, bioconjugation, labeling of living and fixed cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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